CN117568511A

CN117568511A - SNP locus and CAPS molecular marker for identifying early-growth traits of tea trees and application of SNP locus and CAPS molecular marker

Info

Publication number: CN117568511A
Application number: CN202311547487.0A
Authority: CN
Inventors: 刘硕谦; 黄梦迪; 田娜; 苏芹; 刘仲华; 黄建安
Original assignee: Hunan Agricultural University
Current assignee: Hunan Agricultural University
Priority date: 2023-11-20
Filing date: 2023-11-20
Publication date: 2024-02-20

Abstract

The invention provides SNP loci and CAPS molecular markers for identifying early-growth traits of tea trees and application thereof. The SNP locus is positioned at 227558248 base of chromosome 4 of tea tree, the base polymorphism is C/G, and the SNP locus is named as Chr04:227558248_C/G; the primer sequence of CAPS molecular marker converted by SNP locus is primer F:5'-CTGCCAACTTCCCTTATTGTTGTT-3'; primer R:5'-ATGCTCAAGACGCAACAATCTTACT-3'. The method comprises the steps of carrying out PCR amplification by taking genomic DNA of tea trees as a template, carrying out restriction enzyme BstNI digestion on amplification products, carrying out homozygous, heterozygous and wild enzyme digestion on different tea tree varieties through agarose gel electrophoresis, enzyme digestion analysis and the like, wherein 1 band is a homozygous GG type early-matured variety, 3 bands are heterozygous CG type medium-matured varieties, and 2 bands are wild CC type late-matured varieties. The CAPS molecular marker provided by the invention has the advantages of stable band type, high accuracy and low cost, can well distinguish different spring tip germination type tea tree varieties, and can provide a new way for early genotype identification and breeding selection of early tea tree varieties in the future.

Description

SNP locus and CAPS molecular marker for identifying early-growth traits of tea trees and application of SNP locus and CAPS molecular marker

Technical Field

The invention belongs to the technical field of molecular markers, and relates to SNP loci and CAPS molecular markers for identifying early-growth traits of tea trees and application thereof.

Background

Tea tree (Camellia sinensis) is used as a leaf economic plant, spring tip germination time is one of main agronomic characters, and the tea leaves with early germination have better yield and quality, so that the tea leaves have higher economic value.

CAPS molecular markers are a technology that combines PCR technology and restriction cleavage. The marking principle is that mutation of bases on genome fragments can cause change of recognition sites of restriction enzymes, specific primers are designed according to known sequences, PCR amplification is carried out on SNP mutation sites by using the specific primers, the PCR products are digested by specific restriction enzymes, the digested products are detected by agarose gel electrophoresis, the electrophoresis results show that part of genotype amplified fragments can be completely digested into 2 strips, and part of genotype amplified fragments are not digested or are not completely digested into 1 strip or 3 strips, so that single nucleotide polymorphism analysis is carried out. Researches show that the technology has the advantages of high polymorphism, co-dominance, small required DNA amount, simple operation, stable and reliable results and the like.

The current method for identifying early-maturing varieties of tea trees mainly comprises phenotypic selection, and molecular marker assisted selection is less in application, such as simple sequence repeat markers (Simple sequence repeat, SSR). The manual selection of early-maturing varieties of tea trees is time-consuming and labor-consuming, not only depends on the experience of breeders, but also is easily affected by the environment and genotype. This selection is more suitable for selecting simple quality traits, and various studies indicate that tea tree spring tip germination is a quantitative trait controlled by multiple genes. SSR markers are polymorphic but require a large number of primers for developing the markers, and are relatively expensive. With the development of high-throughput sequencing technology, the reduction of sequencing cost enables SNP markers to develop rapidly, and gradually replaces SSR markers. The conversion of SNPs to CAPS markers or dCAPS markers is relatively inexpensive and suitable for routine molecular biology experiments, and has proven to be an effective means of detecting SNPs.

The Chinese patent No. 110016521B discloses a molecular marking method for rapidly identifying germplasm of a bud She Zao green tea tree, wherein SNP (single nucleotide polymorphism) marking sites related to the patent are Sc0002405-P269473 which are closely linked with the She Mengya-stage character of the tea tree bud, a pair of dCAPS marking primers are designed according to DNA sequences near the SNP marking, PCR (polymerase chain reaction) amplification, electrophoretic separation and genotype analysis are carried out on different tea tree germplasm DNA by using the primers, and the germplasm of the bud She Zao green tea tree can be screened out, so that molecular marking auxiliary breeding of the bud She Zao green tea tree variety is realized. The patent compares the electrophoresis pattern of the germplasm resources of the tea tree to be detected with the standard electrophoresis pattern of the germplasm resources of the early-maturing tea tree, if the patterns are consistent, the tea tree is judged to be a bud She Zao raw tea tree variety, but the specific tea tree variety in the provided standard electrophoresis pattern of the germplasm resources of the early-maturing tea tree is not indicated, but the judging accuracy is only 80%, and a larger improvement space is still provided. In addition, the dCAPS molecular labeling method adopted by the method has very small phase difference of enzyme sections, and the conventional electrophoresis is difficult to obtain an ideal separation effect, so that the band type detection is difficult.

Chinese patent No. CN114350847B provides 3 SNP loci for identifying early-maturing tea tree, which are respectively located at 179776419 th locus (base polymorphism is A/G) of a fourth chromosome, 97586343 th locus (base polymorphism is C/T) of a fifth chromosome and 163016524 th locus (base polymorphism is C/T) of a ninth chromosome of Shucha early reference genome. Through genotype analysis of one, two or three of the three SNP loci and screening of samples to be bred according to genotype results, early-maturing tea tree resources can be effectively and rapidly identified. However, the accuracy depends on the type of the site, and a plurality of SNP sites are needed to achieve ideal accuracy, thereby increasing the identification time and cost.

The germination period of tea tree spring tips is a complex character controlled by multiple genes, is easily influenced by environmental conditions, and the screening of tea tree resources with early-growth characters by using molecular markers is a main direction of future tea tree breeding. However, the molecular markers or sites for identifying early-matured tea trees, which can be applied and produced in a large scale, are still lacking so far, and therefore, the development of the molecular markers for tea trees, which are associated with the early-matured state accurately and conveniently, has a wide application prospect.

Disclosure of Invention

Aiming at the problem that the accuracy of the CAPS molecular marker-assisted early-maturing tea tree resource identification is not ideal in the prior art, the SNP locus and the CAPS molecular marker for identifying the early-maturing character of the tea tree are screened by utilizing the whole genome correlation technology, and the molecular marker can be used as the auxiliary identification selection of the early-maturing variety of the tea tree, has high accuracy and simple and convenient operation, and can be applied to large-scale screening of the early-maturing tea tree resource.

In order to achieve the above purpose, the invention adopts the following technical scheme:

one of the purposes of the invention is to provide a SNP locus for identifying the early growth trait of tea trees, wherein the SNP locus is positioned at 227558248 base of chromosome 4 of tea trees, the base polymorphism is C/G, and the SNP locus is named as Chr04:227558248_C/G.

The inventor determines a TGY040711 gene (auxilin-like protein 1) which is highly expressed in spring tips of tea trees on chromosome 4 by carrying out whole genome association analysis on 126 tea tree materials, uses the TGY040711 gene as a candidate gene, uses R software to carry out association analysis on molecular variation data of the gene TGY040711 and spring tip germination phenotype data of tea tree varieties, and screens 1 SNP site (Chr 04: 227558248_C/G) which is obviously associated with early shape of the tea trees in a promoter region of the gene.

The second object of the invention is to provide a CAPS molecular marker for identifying the early-growth traits of tea trees, wherein the nucleotide sequence of the CAPS molecular marker is shown as SEQ ID NO. 1, and the SNP locus is positioned at the 350 th position of the nucleotide sequence.

Experiments prove that when the non-synonymous SNP locus has C base, the amplified fragment can only be digested by BstNI, and when the SNP locus is mutated into G base, the amplified fragment can not be digested by BstNI. The nucleotide sequence of CAPS mark is shown in SEQ ID NO. 1, which is converted into CAPS mark by selecting the base sequence of about 500bp before and after the SNP site of the enzyme cutting site of BstNI enzyme.

It is a third object of the present invention to provide a primer for amplifying the CAPS molecular tag, wherein the primer has the following sequence:

primer F:5'-CTGCCAACTTCCCTTATTGTTGTT-3' (SEQ ID NO: 2);

primer R:5'-ATGCTCAAGACGCAACAATCTTACT-3' (SEQ ID NO: 3).

The fourth object of the present invention is to provide a method for identifying the early-maturing traits of tea trees by using the CAPS molecular markers and primers, comprising the following steps:

(1) Extracting DNA of a tea tree sample to be detected;

(2) Taking DNA of a tea tree sample to be detected as a template, and carrying out PCR amplification by using the primer to obtain an amplification product;

(3) Performing enzyme digestion on an amplification product by using restriction enzyme BstNI, and then performing electrophoresis to obtain an electrophoresis band type of a tea tree sample to be detected;

(4) Band type comparison and identification: if the electrophoresis band type is 2 bands, the genotype of the SNP locus (Chr 04: 227558248_C/G) of the tea tree sample to be detected is CC, and the tea tree sample to be detected is identified as a late-growth variety; if the electrophoresis band type is 3 bands, the genotype of the SNP locus (Chr 04: 227558248_C/G) of the tea tree sample to be detected is CG, and the tea tree sample to be detected is identified as a medium-sized variety; if the electrophoresis band type is 1 band, the genotype of the SNP locus (Chr 04: 227558248_C/G) of the sample of the tea tree to be detected is GG, and the tea tree to be detected is identified as the early-maturing variety.

Further, the reaction system for PCR amplification in each step (2) comprises: 1. Mu.L of primer F, 1. Mu.L of primer R, 1. Mu.L of template DNA, 10. Mu.L of 2X Rapid Taq Master Mix, 7. Mu.L of ddH ₂ O。

Further, the reaction conditions for the PCR amplification in step (2) are: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 40s for 35 cycles; extending at 72 ℃ for 5min; preserving at 4 ℃.

Further, the cleavage system in each step (3) comprises 5. Mu.L of the amplification product, 0.5. Mu.L of the restriction enzyme BstNI, 10X FastDigest Green Buffer. Mu.L, ddH ₂ O8.5 μl; the enzyme cutting conditions are thatIncubate at 37℃for 30min.

Further, when the electrophoresis band type in the step (4) is 2 bands, the sizes of the band fragments are 138bp and 350bp respectively; when the electrophoresis band type is 3 bands, the sizes of the band fragments are 488bp, 138bp and 350bp respectively; when the electrophoresis band type is 1 band, the band fragment size is 488bp.

According to the method for identifying the early-maturing variety of the tea tree, the SNP locus is of a reference genome type, namely, when the SNP locus is of a C allele type only, bstNI is used for enzyme digestion, enzyme digestion can be carried out, and enzyme digestion products are respectively 350bp and 138bp; the variety with genotype CG contains three bands of 350bp, 138bp and 488bp, and the amplified fragment of the variety with G allele type is cut by BstNI, and can not be cut by enzyme, and the band is still 488bp.

The fifth purpose of the invention is to provide and claim the application of the SNP locus, CAPS molecular marker and primer in identifying early-growth traits of tea trees and assisting selective breeding.

It is a sixth object of the present invention to provide and claim the use of the above method in assisted selection breeding.

The invention relates to SNP loci and CAPS molecular markers for identifying early-growth traits of tea trees and application thereof, and belongs to the technical field of molecular markers. The invention finds a candidate gene TGY040711 related to early-growing shape of tea trees based on the whole genome association analysis data of the tea trees, 1 SNP site obviously related to target characters is arranged on the gene, and related CAPS primers are designed for application. Compared with the prior art, the invention has the beneficial effects that:

1. according to the invention, 1 SNP locus (Chr 04: 227558248_C/G) obviously associated with the early-maturing shape of the tea tree is obtained in the promoter region of the TGY040711 gene, and SNP-CAPS molecular markers closely linked with the early-maturing shape are developed, so that the method has higher accuracy in identifying the early-maturing shape of the tea tree, and the accuracy in identifying the early-maturing variety can reach more than 91%.

2. The CAPS molecular marker is utilized to carry out polymorphism verification on 12 tea tree varieties with different spring tip germination traits, and further verify the remaining 72 tea tree germplasm resources, and the result shows that the molecular marker is obviously or extremely obviously associated with the early growth of tea tree. This shows that the CAPS molecular marker of the invention is used for early identification, and has the advantages of convenience, rapidness and high accuracy.

3. The CAPS molecular marker provided by the invention is utilized to carry out genotype identification on SNP loci (Chr 04:227558248_C/G) in 6 different tea tree seedlings, and the spring tip germination phenotype character is cultivated and verified, and the result shows that the genotype of the spring tip germination phenotype character is completely consistent with that of the seedling stage, so that a novel approach can be provided for early identification and selection of breeding materials of the early-maturing character of the tea tree by utilizing the CAPS molecular marker.

4. The invention converts the SNP locus which is screened and early-matured with the tea tree into CAPS mark, which can be completed by simple PCR, restriction enzyme digestion and agarose electrophoresis, and the difference of the sizes of all bands is more than 130bp, thus the invention is easy to identify, and the method has the advantages of high accuracy, rapidness, low cost, short identification period, simple operation and the like. The method can rapidly and effectively evaluate early-maturing of tea trees and assist in selecting tea tree lines with early-maturing.

Drawings

FIG. 1 is a Manhattan plot of a whole genome association analysis of spring tip germination of tea tree.

FIG. 2 is a graph showing the relationship between the mutation site of TGY040711 gene and the early nature of tea tree.

FIG. 3 is a DNA electrophoresis chart of 84 tea plant resources.

FIG. 4 is an electrophoresis detection chart of 12 parts of PCR amplification products of tea plant resources with different spring tip germination characters in example 3, wherein the length of the PCR amplification products is 488bp.

FIG. 5 is a gel electrophoresis chart of 12 parts of PCR amplified products of tea plant resources with different spring tip germination characters in example 3, which are subjected to restriction enzyme BstNI digestion.

FIG. 6 is an electrophoresis detection chart of 12 parts of PCR amplification products of tea plant resources with different spring tip germination characters in comparative example 1, wherein the length of the PCR amplification products is 494bp.

FIG. 7 is a gel electrophoresis chart of 12 parts of PCR amplified products of tea plant resources in comparative example 1, which are digested with restriction enzyme DraI.

FIG. 8 is a gel electrophoresis of 72 parts of PCR amplified product of tea tree germplasm resources cut by restriction enzyme BstNI.

FIG. 9 is a gel electrophoresis of 6 tea tree seedling genomic DNA amplification products digested with restriction enzyme BstNI.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. Unless otherwise indicated, the technical means employed in the examples are conventional means well known to those skilled in the art, and the materials or reagents employed are commercially available or known by published methods.

Example 1 screening and identification of SNP loci of early-stage shape-related genes of tea tree

Analysis of the tea leaf spring shoot germination whole genome correlation data for 126 parts of tea leaf material showed a significant correlation signal on chromosome 4 as shown in fig. 1 (manhattan plot of tea leaf spring shoot germination whole genome correlation analysis), confirming that TGY040711 has a higher expression gene in tea leaf spring shoot germination, thus defining TGY040711 as a candidate gene associated with early shape of tea leaf. The molecular variation data of the gene TGY040711 and the spring tip germination phenotype data of the tea tree variety are subjected to correlation analysis by means of R software, 1 SNP site (Chr 04: 227558248_C/G) (shown in figure 2) which is obviously correlated with the early growth state of the tea tree is screened in a promoter region of the gene, the wild type and mutant nucleotide sequences are compared by DNAMAN7 software, the SNP site is verified, and finally, when the non-synonymous SNP site is found to have C base, an amplified fragment can only be digested by BstNI, and when the SNP is mutated into G base, the SNP can not be digested by BstNI.

Example 2 SNP site (Chr 04: 227558248_C/G) transformation CAPS molecular markers and related primers

Selecting endonuclease BstNI enzyme, selecting base sequences of about 500bp before and after SNP sites of BstNI enzyme cutting sites as CAPS molecular markers, wherein the nucleotide sequence of the CAPS molecular markers is shown as SEQ ID NO. 1, and designing primer sequence pairs of the CAPS molecular markers by utilizing Snapgene software; the information on the labels and their primers used in the positioning process is shown in Table 1.

TABLE 1 SNP site (Chr 04: 227558248_C/G) related CAPS molecular markers, primer sequence information

The marking point of the CAPS molecular marker sequence ((SEQ ID NO: 1)) is an enzyme cutting site of BstNI enzyme, and the bold base is a mutation site.

The primer sequences were sent to the engineering (Shanghai) Co.Ltd.

Example 3CAPS molecular marker (Chr 04:227558248_C/G) enzyme-cleaved band polymorphism and correlation with early-maturing shape verification

1. Tea tree spring tip germination phenotype observation

The field observation was performed on 84 tea tree varieties from a tea base (113°e,28°n) in the hunan province, the spring tip germination phenotype of the tea tree population was recorded using germination index (SPI), the phenotype observation was performed when most of the offspring reached the "one-bud-one-leaf" state, the SPI of each bud was digitally recorded, 0.5 indicating that germination began, 1 indicating one bud, 1.5 indicating one-bud-one-leaf initial expansion, 2 indicating one-bud-one-leaf, 2.5 indicating one-bud-two-leaf initial expansion, 3 indicating one-bud-two-leaf, and so on, and the earlier the germination period of the individual plant with a larger germination value. The first two axillary buds of each robust shoot were selected for SPI scoring, with the SPI for each individual being the average of at least 5-10 overwintering buds.

Tea tree spring tips germination types are roughly classified into 3 types according to SPI values: early-maturing variety (SPI is more than or equal to 3) represented by symbol E; late variety (SPI < 1.9), denoted by symbol L; the Chinese variety (H is more than or equal to 1.9 and less than 3) is represented by a symbol M.

2. Tea tree material to be tested

Picking up one bud and two leaves of the 84 tea tree varieties observed above, fixing the samples with liquid nitrogen, and storing in a refrigerator at-80 ℃.

3. CAPS molecular marker enzyme-digested polymorphism and verification of association with early-maturing state

(1) Extracting DNA of tea tree sample to be detected

84 parts of preserved tea tree materials are all adoptedPlant DNAIsolation Mini Kit (Nanjinouzan Biotechnology Co., ltd.) and the DNA product obtained by extracting the genomic DNA from the kit were subjected to electrophoresis on 1.0% agarose gel to verify the DNA quality, and the results of partial material verification are shown in FIG. 3, and it was found that the genomic DNA of the extracted tea tree was uniform in size and good in quality.

(2) PCR amplification

Selecting 12 tea tree genome DNA with different spring tip germination characters as a template, and performing specific PCR amplification by using CAPS molecular marker primers provided in the example 2 to obtain an amplification product;

the 20. Mu.L PCR amplification reaction system comprises: 1. Mu.L of primer F, 1. Mu.L of primer R, 1. Mu.L of template DNA, 10. Mu.L of 2X Rapid Taq Master Mix, 7. Mu.L of ddH ₂ O。

The reaction conditions for PCR amplification were: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 40s for 35 cycles; extending at 72℃for 5min.

Taking 4 mu L of PCR amplified product, detecting the amplified result (M, marker D2000) on 1.0% agarose gel by electrophoresis, confirming that the amplified result is accurate, and storing at 4 ℃ for standby. As shown in FIG. 4, the sizes of the amplified product band fragments obtained by taking the 12 tea tree genome DNA as a template are consistent, and the length of the PCR amplified product is 488bp.

(3) Enzyme cutting

Enzyme cutting the amplification products of the 12 different tea tree varieties genome DNA by using restriction enzyme BstNI, respectively carrying out electrophoresis on the obtained enzyme cutting products on 1.0% agarose gel, and then observing electrophoresis bands on a gel imager to preserve experimental results;

15 mu L enzyme digestionThe system comprises 5. Mu.L of amplified product, 0.5. Mu.L of restriction enzyme BstNI, 10X FastDigest Green Buffer. Mu.L, ddH ₂ O8.5 μl; the digestion conditions were 37℃for 30min.

(4) Analysis of results

FIG. 5 is an electrophoresis chart of an enzyme-sectioned region obtained by CAPS molecular marker detection of 12 varieties with different spring tip germination traits (early-maturing traits) in the embodiment, and simultaneously, the spring tip germination types of 12 selected tea plant materials are marked on lanes corresponding to the electrophoresis chart, as shown in FIG. 5, and the electrophoresis chart comprises 2 parts of early-maturing varieties (lanes 1 and 11), 6 parts of middle-maturing varieties (lanes 4-8 and 12) and 4 parts of late-maturing varieties (lanes 2, 3, 9 and 10).

As shown in FIG. 5, 3 types of bands, 1 band (488 bp in length), 3 bands (488 bp, 138bp and 350bp in length), and 2 bands (138 bp and 350bp in length), respectively, appeared in the electrophoretogram. 2 tea plant materials corresponding to 1 band are early-maturing varieties (E), 5 tea plant materials corresponding to 3 bands are medium-maturing varieties (M), and 5 tea plant materials corresponding to 2 bands are 4 late-maturing varieties (L) and 1 medium-maturing varieties (M), so that the enzyme-cutting polymorphism of the molecular marker (Chr 04: 227558248_C/G) is better, the enzyme-cutting band type has good corresponding relation with the spring tip germination phenotype of the tea tree, the early-maturing varieties (E) correspond to 1 band, the medium-maturing varieties (M) correspond to 2 bands, the late-maturing varieties (L) correspond to 2 bands, and the accuracy is about 91.7%, and the early-maturing varieties, the medium-maturing varieties and the late-maturing varieties of the tea plant can be effectively identified by using the CAPS molecular marker method.

Comparative example 1 screening and detection of another SNP site of early-growth-related Gene TGY040711, CAPS marker

1. Screening of another SNP site

In the screening and identifying process of SNP locus in example 1, TGY040711 is also used as a candidate gene related to early shape of tea tree, and when molecular variation data of gene TGY040711 and spring tip germination phenotype data of tea tree variety are subjected to correlation analysis by means of R software, another SNP locus (Chr 04: 2275585569_A/T) which is obviously related to early shape of tea tree is screened, as shown in FIG. 2; and comparing the wild type nucleotide sequence and the mutant nucleotide sequence by DNAMAN7 software, verifying the SNP locus, and finally finding that when the SNP locus has an A base, the amplified fragment can only be digested by DraI, and when the SNP is mutated into a T base, the amplified fragment can not be digested by DraI.

2. SNP site (Chr 04: 2275585569_A/T) transformation CAPS marker and related primers

Selecting endonuclease DraI enzyme, selecting base sequences of about 500bp before and after SNP sites positioned at enzyme cutting sites of DraI as CAPS molecular markers, wherein the nucleotide sequence of the CAPS molecular markers is shown as SEQ ID NO. 4, and designing primer sequence pairs of the CAPS molecular markers by utilizing Snapgene software; the information on the labels and their primers used in the positioning process is shown in Table 2.

TABLE 2 SNP site (Chr 04: 2275585569_A/T) related CAPS molecular markers, primer sequence information

The marking point of the CAPS molecular marker sequence (SEQ ID NO: 4) is an enzyme cutting site of DraI enzyme, and the bold base is a mutation site.

The primer sequences were sent to the engineering (Shanghai) Co.Ltd.

3. Detection and verification of spring tip germination type of tea tree by CAPS molecular marker (Chr 04:227558559_A/T)

A variety with 12 spring tips having different germination traits was selected as in example 3.

The selected 12 varieties were sequentially subjected to amplification, cleavage, and electrophoresis according to the detection method described in example 3, wherein PCR amplification was performed using the primers (SEQ ID NO:5, SEQ ID NO: 6) shown in Table 2, cleavage was performed using DraI enzyme, and the other conditions were kept identical.

The electrophoresis result of the PCR amplification products of 12 varieties is shown in FIG. 6, and the band size of the PCR amplification products is 494bp; the result of electrophoresis of the enzyme-sectioned fragments obtained by detecting 12 parts of varieties by CAPS molecular markers (Chr 04: 2275555959_A/T) is shown in FIG. 7, and the spring tip germination types of 12 parts of tea plant materials are marked on lanes corresponding to the electrophoresis patterns as in example 3, and the electrophoresis comprises 2 parts of early-maturing varieties (lanes 1 and 11), 6 parts of medium-maturing varieties (lanes 4-8 and 12) and 4 parts of late-maturing varieties (lanes 2, 3, 9 and 10).

As shown in figure 7, after comparison and analysis of 12 tea tree varieties with corresponding marked tea tree spring tips germination types, the enzyme-cut products have a certain polymorphism, and 2 bands and 3 bands appear, but the degree of coincidence with the tea tree spring tips germination characters is lower, and the accuracy is only 41% -50%.

Example 4 further verification of the relationship of tea Tree germplasm resources enzyme-cut bands and tea Tree early-growth shapes

In the embodiment, the CAPS molecular marker (Chr 04: 227558248_C/G), the primer F and the primer R provided in the embodiment 2 are utilized, the correlation condition of the enzyme-digested bands of the remaining 72 tea plant germplasm resources and the early-maturing shape (spring tip germination type) in the embodiment 3 is further verified by the verification method provided in the embodiment 3, and the reliability of the detection result is verified by analyzing the relation between SNP loci (Chr 04: 227558248_C/G) and the early-maturing shape of the tea plant under different genetic models.

1. Tea tree material to be tested

In example 3, the remaining 72 parts of tea plant material were stored in a-80℃refrigerator after solidification with liquid nitrogen.

2. Detection method

The procedure was identical to the detection method in example 3.

3. Analysis of results

FIG. 8 (A-C) shows an electrophoresis pattern of enzyme-sectioned fragments obtained by detecting 72 parts of tea plant germplasm resources through CAPS molecular markers (Chr 04:227558248_C/G) in the embodiment, wherein the spring tip germination types of 72 parts of tea plant germplasm resources comprise 14 parts of early-maturing varieties (E), 28 parts of medium-maturing varieties (M) and 30 parts of late-maturing varieties (L), the corresponding markers of the varieties are marked with lanes, and the electrophoresis band types of the enzyme-sectioned fragments are compared with the spring tip germination types, so that the correlation conditions found in the embodiment 3 are consistent, and the accuracy rate can reach more than 95.8%.

And counting genotypes of 72 tea tree materials, carrying out correlation analysis on enzyme-cut banding and tea tree spring shoot germination types by using R software according to a counting result under different genetic models, and showing that the locus genotypes are obviously or extremely obviously correlated with tea tree spring shoot germination phenotype traits. As shown in Table 3, under the co-dominant model, the C/C genotype of the Chr04:227558248_C/G locus was very significantly associated with early tea plant growth (P < 0.01); the C/C genotype of the Chr04:227558248_C/G locus is extremely significantly associated with early tea plant shape in dominant model (P < 0.01); the C/C and C/G genotypes at the Chr04:227558248_C/G locus are significantly associated with the early vigour of tea trees (P < 0.05) under a recessive model. Under the super dominant model, the C/C and G/G genotypes of the Chr04:227558248_C/G locus are extremely significantly associated with the early shape of tea trees (P < 0.01).

TABLE 3 relation of mutation sites to tea Tree spring tip germination type (early growth trait) under different genetic models

Example 5 identification and verification of early-maturing status of tea seedlings

6 mature tea tree seeds are randomly selected for germination, one tea tree seedling with one week germination is taken as a material, a tender leaf (about 0.2g in wet weight) is taken as each material, the CAPS molecular marker and the primer provided in example 2 and the verification method provided in example 3 are adopted for early-stage identification of the tea tree seedlings, the electrophoresis patterns of enzyme sections obtained by detecting 6 tea tree seedlings through the CAPS molecular marker are shown in fig. 6, and the electrophoresis patterns are respectively 1 band, 2 band, 3 band, 2 band and 3 band, so that the genotypes are respectively homozygous mutant (GG), wild type (CC), heterozygous mutant (CG), wild type (CC) and heterozygous mutant (CG).

The method is as described in example 3, and the result shows that 6 parts of materials can be respectively identified as early-maturing variety, late-maturing variety, medium-maturing variety, late-maturing variety and medium-maturing variety according to SPI value, and the genotypes of the 6 parts of materials are completely consistent with those of the young seedling stage (shown in figure 6). Therefore, the molecular marker can accurately predict the early-maturing character of the tea tree in the seedling stage, greatly shortens the period of breeding early-maturing tea tree varieties, and can be applied to large-scale production.

According to the results and analysis of the embodiment, the SNP locus and CAPS molecular marker (comprising a primer and a restriction enzyme) provided by the invention can effectively identify the early-maturing characters of different tea plant resources and divide the early-maturing characters into three spring tip germination types of early-maturing varieties, medium-maturing varieties and late-maturing varieties. The method is rapid and effective, can be used for identification in the seedling stage, and provides assistance for the breeding selection work of tea tree varieties.

The foregoing examples are set forth in order to provide a more thorough description of the present invention, and are not intended to limit the scope of the invention, since modifications of the invention in various equivalent forms will occur to those skilled in the art upon reading the present invention, and are within the scope of the invention as defined in the appended claims.

Claims

1. A SNP locus for identifying the early growth character of tea trees is characterized in that the SNP locus is positioned at 227558248 base of chromosome 4 of tea trees, the base polymorphism is C/G, and the SNP locus is named as Chr04:227558248_C/G.

2. A CAPS molecular marker for identifying early-growth traits of tea trees is characterized in that the nucleotide sequence of the CAPS molecular marker is shown as SEQ ID NO. 1, and the nucleotide sequence comprises the SNP locus of claim 1.

3. Amplifying the CAPS molecular tagged primer of claim 2, wherein the primer has the sequence:

primer F:5'-CTGCCAACTTCCCTTATTGTTGTT-3';

primer R:5'-ATGCTCAAGACGCAACAATCTTACT-3'.

4. A method for identifying the early-growth traits of tea trees by using CAPS molecular markers according to claim 2, comprising the following steps:

(1) Extracting DNA of a tea tree sample to be detected;

(2) Carrying out PCR amplification by using the primer of claim 3 by taking DNA of a tea tree sample to be detected as a template to obtain an amplification product;

(3) Enzyme-cutting an amplification product by using restriction enzyme BstNI, and then electrophoresis to obtain an electrophoresis band type of the tea tree sample;

5. The method according to claim 4, wherein the reaction system for PCR amplification in each step (2) comprises: 1. Mu.L of primer F, 1. Mu.L of primer R, 1. Mu.L of template DNA, 10. Mu.L of 2X Rapid Taq Master Mix, 7. Mu.L of ddH ₂ O。

6. The method of claim 4, wherein the reaction conditions for the PCR amplification in step (2) are: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 40s for 35 cycles; extending at 72 ℃ for 5min; 4. preserving at the temperature.

7. The method according to claim 4, wherein the cleavage system in each step (3) comprises 5. Mu.L of the amplification product, 0.5. Mu.L of the restriction enzyme BstNI, 10X FastDigest Green Buffer. Mu.L, and ddH ₂ O8.5 μl; the digestion conditions were 37℃for 30min.

8. The method according to claim 4, wherein in the step (4), when the electrophoresis band type is 2 bands, the sizes of the band fragments are 138bp and 350bp, respectively; when the electrophoresis band type is 3 bands, the sizes of the band fragments are 488bp, 138bp and 350bp respectively; when the electrophoresis band type is 1 band, the band fragment size is 488bp.

9. Use of the SNP locus according to claim 1, CAPS molecular marker according to claim 2, the primer according to claim 3 for identifying tea tree early-growth traits and assisting in selective breeding.

10. Use of the method of any one of claims 4-8 in assisted selection breeding.